Coxiella burnetii is an obligate intracellular bacterial pathogen and the etiologic agent of human Q fever. Q fever typically presents as an acute debilitating flu-like illness but can progress to severe, chronic endocarditis. In host cells, C. burnetii establishes a replicative niche in a lysosome-like parasitophorous vacuole. While discovered over 70 years ago, our understanding of mechanisms used by the pathogen to infect host cells and cause disease is still fragmentary, specifically with respect to the identity and activity of discrete virulence factors. During intracellular growth, C. burnetii actively manipulates multiple host processes, including vesicular trafficking and cell survival, to establish an efficient infection. However, the bacterial proteins mediating these events are not known. The major mode of secretion used by C. burnetii to deliver bacterial proteins to the host cell appears to be the Dot/lcm type IV secretion system. The current proposal is designed to functionally characterize C. burnetii proteins delivered to the host cytosol during infection via the Dot/lcm type IV secretion system. To this end, Specific Aim 1 is designed to characterize the temporal expression of Dot/lcm substrates that we have already identified and investigate host proteins that interact with these effectors. These studies will involve the use of RT-PCR and co- immunoprecipitation approaches to elucidate effector activity during infection and intracellular growth. Specific Aim 2 will use three approaches to characterize Dot/lcm substrate function during C. burnetii infection. First, an siRNA approach will be used to knockdown expression of effector binding host proteins and analyze the effect of this loss on C. burnetii infection and replication. Second, individual effectors will be examined for their role in subversion of host signaling, including inhibition of apoptosis and activation of the pro-survival proteins Akt, Erk1/2, and NF-icB. Third, individual effectors will be targeted with specific polyclonal antiserum during infection to investigate their role in vesicular fusion events required for proper trafficking of the pathogen-containing vacuole. Collectively, the proposed studies will identify novel C. burnetii virulence determinants and provide an enhanced understanding of mechanisms used by C. burnetii to efficiently parasitize host cells. Additionally, these studies will further our understanding of basic host cell signaling processes and the interaction of intracellular pathogens with the host. RELEVANCE (See instructions): C. burnetii is a CDC Category B select agent and causes the zoonosis human Q fever, a debilitating acute disease that also presents as chronic endocarditis. Characterization of C. burnetii Dot/lcm effectors will identify candidate proteins for vaccine components and therapeutics to combat this disease and will provide insight into the complex interactions between this pathogen and the host.